Research On The Influence Of Heterologous Protein Expressed By Pichia Pastoris By Cystatin And Its Mutant As Examples

Pichia pastoris is a highly efficient eukaryotic expression system that has been widely used to produce recombinant proteins.Existing studies have shown that over-expression of the Protein Disulfide Isomerase(PDI) in yeasts can significantly increase the expression level of foreign proteins.PDI is also involved in protein folding while catalyzing the formation of nascent protein disulfide bonds;On the other hand,it has the function of a molecular chaperone,and can help to fold the folded protein into its natural conformation.In order to analyze the mechanism of PDI to increase the efficiency of amyloid expression,the cellular pathways and signal factors,chicken cystatin was used as a model protein and various mutants were constructed,including amyloid mutant I66 Q,the unstable mutant ?W and the ?-helix II disintegration mutant E82 P,these mutants were secreted in the PDI normal expression and overexpressing Pichia pastoris strain GS115.The intracellular and extracellular proteins of each strain were detected by SDS-PAGE and Western Blot.The results showed that two recombinant strains expressing of I66 Q wasn't detected the intracellular protein,and the other strains was detected the monomer of the foreign protein in the cells.The results of extracellular protein showed that the expression levels of PDI overexpressing strains were significantly higher than those of PDI normal expression strains except that the expression level of E82 P in PDI normal expression and overexpression strains was not significantly changed.The experimental results of protein expression are consistent with the existing scientific hypothesis that protein conformation determines expression efficiency.The strains were analyzed by transcriptome sequencing technology.Venn diagrams were generated for the two strains of I66 Q VS WT and ?W VS WT,and 203 and 210 differentially expressed genes were screened.The KEGG pathways of these differentially expressed genes were classified and counted.It was found that 145 and 148 genes were annotated into KEGG pathways in I66 Q VS WT and ?W VS WT groups,respectively.There was a significant difference in genetic information processing of differentially expressed genes.Especially in the process of DNA replication,only gene 3858 was up-regulated in I66 Q VS WT,while five genes were up-regulated in ?W VS WT in DNA replication pathway.Therefore,we speculate that the reason for such a large gap is that although I66 Q is an amyloid mutant,its main conformation changes little compared with the wild type,while ?W not only removes an ?-helix II containing nine amino acids but also lacks a disulfide bond,thus requiring induction of more genes to improve the efficiency of its functions related to protein synthesis such as replication,transcription and translation.The results of this study and transcriptome sequencing analysis confirmed the regulation of PDI on the secretion of amyloid by Pichia pastoris.On the other hand,comparing the similarities and differences between chicken cystatin mutants and wild-type chicken cystatin,it is helpful to elucidate the intracellular production process of amyloid mutants,unstable mutants and wild-type natural proteins of nascent proteins.Provide theoretical and experimental basis for understanding the quality management system of eukaryotes.